Interleukin-5 (IL-5) is a lymphokine secreted by activated T cells which is biologically active on B cells and eosinophils. Because IL-5 replaces T lymphocytes in in vitro antibody responses to thymus-dependent antigens, it was formerly called T cell replacing factor [TRF; Dutton et al., Prog. Immunol. 1:355 (1971); Schimpl et al., Nature 237:15 (1972)]. Because it also stimulates differentiation of B lymphocytes into IgM and IgG plaque-forming cells and the growth of B cell lymphomas in vitro, it has also been called B cell growth factor II [BCGFII; Takatsu et al., J Immunol. 124:2414 (1980)].
Murine IL-5 consists of 133 amino acid residues, including a signal sequence of 20 residues and three potential N-glycosylation sites. Deglycosylation does not affect the biological activity of murine IL-5 in a B cell proliferation assay [Tavernier et al., DNA 8:491 (1989)]. Human IL-5 consists of 134 amino acid residues, including a signal sequence of 19 residues and two potential N-glycosylation sites. The structures of both proteins have been described by Yokota et al. [Proc. Natl. Acad. Sci. USA 84:7388 (1987)] and Kinashi et al. [Nature 324:70 (1986)]. The degrees of homology of murine and human IL-5 at the nucleotide and amino acid sequence level are 77 and 70%, respectively.
Both murine and human IL-5 exist as homodimers linked by disulfide bonds. Therefore, glycosylated recombinant human IL-5 migrates in SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 40,000 daltons under non-reducing conditions, and 20-22,000 daltons under reducing conditions [Tsujimoto et al., J. Biochem. 106:23 (1989)].
The cloning and expression of murine IL-5 has been described, e.g., by Kinashi et al. [Nature 324:70 (1986)] and Takatsu et al. [J. Immunol. 134:382 (1985)]. Human IL-5 complementary DNA (cDNA) has been isolated using murine IL-5 cDNA as a probe by Azuma et al. [Nucleic Acids Res. 14:9149 (1986)].
IL-5 has been shown to act as a maintenance and differentiation factor for eosinophils. In humans, the activity of IL-5 appears to be specific, affecting eosinophils primarily. Human IL-5 induces eosinophil precursor cells to become mature cells. Moreover, the survival of eosinophils isolated from circulating blood can be prolonged when human IL-5 is present in the culture media. Human IL-5 also stimulates cultured eosinophils to degranulate, and to release toxic proteins such as major basic protein (MBP) and eosinophil-derived neurotoxin (EDN) [Kita et al., J. Immunol. 149:629 (1992)].
It has been suggested that eosinophils kill parasites following infection and also play a significant role in inflammatory and allergic diseases [see, e.g., Sanderson, Blood 79:3101 (1992)]. Increased levels of eosinophils among circulating leukocytes have been observed following parasitic infections and in certain chronic inflammatory tissues, such as in asthmatic alveoli. Eosinophil infiltration and toxic granule release from eosinophils may play a role in tissue destruction and may aggravate the symptoms of asthma.
For example, Gleich et al. [Adv. Immunol. 39:177 (1986)] and Frigas et al. [J. Allergy Clin. Immunol. 77:527 (1986)] have shown that high-density eosinophils and eosinophil major basic protein (MBP) are associated with bronchial asthma and related tissue damage.
Recently, Coffman et al. (International Patent Application Publication No. WO90/04979) have shown that antibodies against IL-5 can prevent or reduce eosinophilia which is associated with certain allergic diseases such as asthma. Monoclonal antibodies which specifically bind to and neutralize the biological activity of human IL-5 can be used for this purpose.
A monoclonal antibody against IL-5 has been reported to have a prominent effect in reversing parasite-induced eosinophilia in experimental animals [Schumacher et al, J. Immunol. 141:1576 (1988); Coffman et. al., Science 245:308 (1989)], suggesting that neutralizing antibodies may be clinically useful in relieving eosinophilia-related symptoms by antagonizing IL-5. In fact, it has been reported that when rodents or monkeys bearing experimentally induced eosinophilia were treated with TRFK 5, a rat anti-mouse IL-5 monoclonal antibody, eosinophil counts in both circulation and bronchial lavage were found to return to normal levels. Thus, neutralizing monoclonal antibodies may be effective antagonists.
Because most monoclonal antibodies are of rodent origin, however, there is an increased likelihood that they would be immunogenic if used therapeutically in a human being, particularly over a long period of time. To reduce this possibility, there is a need for recombinant or "humanized" antibodies against human IL-5. Such antibodies could be used for the treatment of conditions associated with eosinophilia, or for the treatment of any other condition attributable to the biological activity of IL-5.
Initial efforts to reduce the immunogenicity of rodent antibodies involved the production of chimeric antibodies, in which mouse variable regions were fused with human constant regions [Liu et al., Proc. Natl. Acad. Sci. USA 84:3439 (1987)]. It has been shown, however, that mice injected with hybrids of human variable regions and mouse constant regions develop a strong anti-antibody response directed against the human variable region. This suggests that in the human system, retention of the entire rodent Fv region in such chimeric antibodies may still give rise to human anti-mouse antibodies.
It is generally believed that CDR loops of variable domains comprise the binding site of antibody molecules, the grafting of rodent CDR loops onto human frameworks (i.e., humanization) was attempted to further minimize rodent sequences [Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)]. Studies by Kabat et al. [J. Immunol. 147:1709 (1991)] have shown that framework residues of antibody variable domains are involved in CDR loop support. It has also been found that changes in framework support residues in humanized antibodies may be required to preserve antigen binding affinity. The use of CDR grafting and framework residue preservation in a number of humanized antibody constructs has been reported, e.g., by Queen et al. [Proc. Natl. Acad. Sci. USA 86:10029 (1989)], Gorman et al. [Proc. Natl. Acad. Sci. USA 88:4181 (1991)] and Hodgson [Bio/Technology 9:421 (1991)]. Exact sequence information has been reported for only a few humanized constructs.
Although a high degree of sequence identity between human and animal antibodies has been known to be important in selecting human antibody sequences for humanization, most prior studies have used a different human sequence for animal light and heavy variable sequences. Sequences of known antibodies have been used or, more typically, those of antibodies having known X-ray structures, antibodies NEW and KOL. See, e.g., Jones et al., supra; Verhoeyen et al., supra; and Gorman et al., supra.
Methods for engineering antibodies have been described, e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S. Pat. No. 4,816,567), Law et al. (European Patent Application Publication No. 438 310) and Winter (European Patent Application Publication No. 239 400).
Reliance on the relatively few antibodies for which X-ray structures have been determined has led to the frequent use of different human light and heavy chain sequences from different antibodies, because although only two human Fab crystal structures are known, several human light chain crystal structures have been determined. Such an approach may require changing framework residues in the human heavy and light chains to ensure correct chain association and, therefore, limits the applicability of humanization.
There thus is a need for improved methods for making humanized antibodies that are not based upon the relatively few known crystallographic structures.